Background
In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and quantitation
Conclusions
Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA.
Methods
In this study, we provided a possible explanation for the reduced sensitivity of existing quantitation methods for dsRNA with modified uridines by characterizing the binding affinities of commonly used anti-dsRNA antibodies. Then, a rapid and sensitive biolayer interferometry (BLI) dsRNA detection assay utilizing Flock House Virus (FHV) B2 protein was developed to overcome the challenges in dsRNA detection and the reduced sensitivity.
Results
This assay allows the detection of dsRNA with different uridine modifications (ψ, m1ψ, 5 moU) with similar sensitivity as dsRNA without modification. Furthermore, we demonstrated this method can be used to quantify both short and long dsRNA, as well as hairpin-structured dsRNA, providing a more comprehensive detection for dsRNA impurities. Moreover, we applied this assay to monitor dsRNA removal through a purification process. Conclusions: Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA.